Diabetes is a major cause of morbidity and mortality in the U.S. Given that the diabetes epidemic continues to grow worldwide, there is a clear need for improvements in the management of the disease and its complications. Early diagnosis and intervention can significantly improve long-term prognosis. Autoantibodies against islet antigens are a serologic hallmark of patients with Type 1 Diabetes (T1D). In addition, circulating microRNAs (miRNAs) have been shown recently to be systematically altered, indicative of miRNA signatures with diagnostic utility. Growing evidence suggests that a multi-marker strategy, containing a combination of biomarkers with high clinical sensitivity and specificity, may enhance diagnostic and prognostic accuracy in the future compared to single marker tests. To support efforts of identifying the most informative biomarker panels, reliable next- generation platform technologies are needed that permit multiplexed detection of markers in small samples and are suitable for automation and integration into the clinical laboratory work flow. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for an ultrasensitive and - specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with high single-well multiplexing potential, minimal sample requirements, and simplified handling procedures (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers). NTI recently achieved extension from 2-color to 4-color ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for direct protein as well as miRNA quantification. Furthermore, recent work by Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, termed single molecule optofluidics, and ii) enhanced throughput using a multifoci excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed, ultrasensitive and -specific, as well as fully automated, nucleic acid- and protein-based diagnostic tests that require minimal sample sizes. Here, we propose assay development and clinical validation of a next-generation test with significantly improved diagnostic, prognostic, and treatment- guiding properties, implementing a panel of autoantibody and miRNA biomarkers, and overcoming limitations of current T1D testing. Our Specific Aims are: 1. Initial reagent development for a multiplex autoantibody- and miRNA-based next-generation test for T1D 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of 200 archived clinical samples and cross-validation to ELISA and qPCR methods SBIR Phase II will propose assay expansion to include more markers, miniaturization, and development of a user-friendly, sample in - answer out diagnostic system offering significant cost and patient sample savings.